Haynes Lab:Notebook/CRISPR Editing/2016/02/29: Difference between revisions
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Rene M Davis (talk | contribs) |
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Should have no bands in the gel and bands on the nicrocellulose. Rinse the nitrocellulose in DI water. Move the nitrocellulose to a smaller dish. Pour Ponceau stain over to just cover it. Orbital shake gently for 5-10 minutes. Rinse the Ponceau in the sink in DI water until the background is white.<br> | Should have no bands in the gel and bands on the nicrocellulose. Rinse the nitrocellulose in DI water. Move the nitrocellulose to a smaller dish. Pour Ponceau stain over to just cover it. Orbital shake gently for 5-10 minutes. Rinse the Ponceau in the sink in DI water until the background is white.<br> | ||
Image the nitrocellulose on the syngene PXi machine. Remove the UV box and replace with the black background piece. Blots>visible blot>Ponceau red> -> or used saved protocols "ponceau stain". Keep nitrocellulose in water until time to image. Keep water nearby so you can quickly transfer. Need to make sure it doesn't dry out. Take an image.<br> | Image the nitrocellulose on the syngene PXi machine. Remove the UV box and replace with the black background piece. Blots>visible blot>Ponceau red> -> or used saved protocols "ponceau stain". Keep nitrocellulose in water until time to image. Keep water nearby so you can quickly transfer. Need to make sure it doesn't dry out. Take an image.<br> | ||
[[Image:Screenshot 2016-03-12 20.40.35.png]]<br> | |||
Cut the nitrocellulose to separate the pieces that will be bound with different antibodies. add notches to one corner so you remember the orientation<br> | Cut the nitrocellulose to separate the pieces that will be bound with different antibodies. add notches to one corner so you remember the orientation<br> | ||
To block the nitrocellulose, move to a new dish and pour 5% BSA, 1% tween, 1% PBS over. Ok to stack the nitrocellulose. Keep still at RT for one hour (went about 1.5hours)<br><br> | To block the nitrocellulose, move to a new dish and pour 5% BSA, 1% tween, 1% PBS over. Ok to stack the nitrocellulose. Keep still at RT for one hour (went about 1.5hours)<br><br> |
Revision as of 20:42, 12 March 2016
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02/29/2015Plated Luc14s in 12-well plate, 0.1x10^6 cells/well, for transfections tomorrow
insert calculations for 1:2 ratio
Mixed gently, inc at RT for 30 mins
Ligation for MV14
Insert
Mixed gently, inc at RT for 30 mins Running gel for western Gel lanes
Mastermix for sample buffer
heat at 100degC for 5 mins, cool in rack at RT
remove the strip from the gel, put premade gel into box with the open part facing the outer chamber, fill inner chamber to cover the comb, outer chamber to cover the bottom of the gel.
run gel for 1 hour at 120V (until lowest dye band ran just past the third ridge in gel box)
Cut the nitrocellulose to separate the pieces that will be bound with different antibodies. add notches to one corner so you remember the orientation
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